Article Taxonomy and Molecular Phylogeny of Phellodon (Thelephorales) with Descriptions of Four New Species from Southwest China Chang-Ge Song 1, Xing Ji 1, Shun Liu 1, Xiao-Lan He 2 and Bao-Kai Cui 1,* 1 Institute of Microbiology, School of Ecology and Nature Conservation, Beijing Forestry University, Beijing 100083, China; [email protected] (C.-G.S.); [email protected] (X.J.); [email protected] (S.L.) 2 Soil and Fertilizer Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China; [email protected] * Correspondence: [email protected]; Tel./Fax: +86-10-6233-6309 Abstract: Phellodon is a genus of ectomycorrhizal fungi belonging to the group known as the stipitate hydnoids. It is associated with coniferous trees in forest ecosystems and is widely distributed in the northern hemisphere. Phellodon, together with Hydnellum, and Sarcodon, is classified in the Bankeraceae, members of which are generally considered as symbiotic fungi. Ectomycorrhizal fungi can help plant roots fix nitrogen and improve the absorption capacity of soil nutrients by trees, so they play an important role in ecosystem protection. Taxonomic and phylogenetic studies of Chinese Phellodon collections were carried out. Four new Phellodon species were discovered from southwestern China based on a combination of morphological characters and molecular data. Phellodon atroardesiacus is characterized by the blackish blue to dark grey pileus, dark grey to ash grey spines, and presence of clamp connections in spines. Phellodon cinereofuscus is distinguished by Citation: Song, C.-G.; Ji, X.; Liu, S.; a cottony tomentose pileal margin, long spines which become clay-buff when dry, and echinulate He, X.-L.; Cui, B.-K. Taxonomy and basidiospores. Phellodon stramineus is characterized by a depressed and tomentose pileus, straw Molecular Phylogeny of Phellodon buff-colored pileal surface, and dark grey to ash grey spines. Phellodon yunnanensis is distinguished (Thelephorales) with Descriptions of by a clay-pink to brown pileus, pale brown to white spines, and the presence of clamp connections in Four New Species from Southwest the outer layer of stipe. Detailed descriptions, illustrations, and ecological traits for the new taxa are China. Forests 2021, 12, 932. https:// provided. Phylogenetic analyses inferred from the internal transcribed spacer (ITS) regions confirmed doi.org/10.3390/f12070932 that the four new species are distinct within Phellodon. Academic Editor: Artur Alves Keywords: ectomycorrhizal fungi; species identification; stipitate hydnoid fungi; taxonomy Received: 10 June 2021 Accepted: 14 July 2021 Published: 16 July 2021 1. Introduction Publisher’s Note: MDPI stays neutral Phellodon P. Karst. is a genus of ectomycorrhizal fungi associated with conifer trees in with regard to jurisdictional claims in forest ecosystems. The genus Phellodon, together with Hydnellum P. Karst., and Sarcodon published maps and institutional affil- Quél. ex P. Karst., belongs to stipitate hydnoids, all of which are classified in the family iations. Bankeraceae. Ectomycorrhizal fungi are an important bridge between plant roots and soil and have ecological functions such as improving the absorption capacity of trees to soil nutrients [1]. Ectomycorrhizal fungal agents can also be widely used in seedling breeding of trees, exsitu Copyright: © 2021 by the authors. protection of tree species, restoration and reconstruction of damaged ecosystems, and other Licensee MDPI, Basel, Switzerland. processes [2]. Stipitate hydnoids are the emphasis of conservation in Europe because of This article is an open access article their declining numbers [3]. Many British stipitate hydnoids species (14 species) were distributed under the terms and included in the UK Biodiversity Action Plan as priority species [4]. conditions of the Creative Commons Phellodon was established by Petter Adolf Karsten and its type species is P. niger (Fr.) Attribution (CC BY) license (https:// P. Karst [5]. According to the modern definition, species in Phellodon are characterized by creativecommons.org/licenses/by/ the basidiomata consisting of a stipe and pileus with hydnoid hymenophores, uniform to 4.0/). Forests 2021, 12, 932. https://doi.org/10.3390/f12070932 https://www.mdpi.com/journal/forests Forests 2021, 12, 932 2 of 17 duplex context, hyaline and echinulate basidiospores [3]. Due to indeterminate growth, basidiomata of Phellodon are confluent and acquire irregular shape [6]. Around 18 species have been described in the genus according to He, et al. [7]. Most of these species were recorded from North America [6]. In the 20th century, species of Phellodon were described based only on morphological characteristics [8–15]. In recent years, molecular studies have been used to infer species limits in Phellodon. Parfitt et al. [3] combined morphological methods with DNA sequencing of the ITS1 region to clear the classification status of the known Phellodon species from the UK, which revealed more terminal clusters than conventionally recognized taxa. Baird et al. [7] conducted a study to reevaluate the species of stipitate hydnoid fungi from temperate southeastern United States; species of Phellodon were recorded and Bankera fuligineoalba (J.C. Schmidt) Pouzar was recombined in Phellodon. Then, they discovered a new species, P. mississippiensis R.E. Baird, L.E. Wallace & G. Baker, from the southern United States, which was observed to have rare clamp connections in the subhymenial hyphae [16]. The taxonomy and phylogeny of Phellodon are not well studied from China, and only one species, P. subconfluens H.S. Yuan & F. Wu in Liaoning Province, was recently described by Mu, et al. based on morphological characters and molecular data [17]. During investigations on macrofungi from Yunnan Province, southwestern China, some specimens with stipitate hydnoid basidiomata were collected. Morphological char- acters and phylogenetic analyses based on the internal transcribed spacer (ITS) regions indicated that these specimens represented four undescribed species of Phellodon. The aims of this study are to confirm the taxonomic affinities of the new species, explore the species diversity of Phellodon in southwestern China, and infer the evolutionary relationships among representative species of Phellodon. 2. Materials and Methods 2.1. Morphological Studies The studied specimens were deposited at the herbarium of the Institute of Microbi- ology, Beijing Forestry University (BJFC). Macromorphological descriptions were based on field notes and herbarium specimens. Three specimens were examined of each of the 4 suspected new species, and 30 spores were counted per specimen. Microscopic characters, measurements, and drawings were made from slide preparations stained with Cotton Blue and Melzer’s reagent and observed at magnifications up to ×1000 under a light microscope (Nikon Eclipse E 80i microscope, Nikon, Tokyo, Japan) following Sun, et al. [18] and Han, et al. [19]. In the text, the following abbreviations were used: IKI = Melzer’s reagent, IKI– = negative in Melzer’s reagent, KOH = 5% potassium hydroxide, CB = Cotton Blue, CB+ = cyanophilous, CB− = acyanophilous, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q = variation in the L/W rationes between the specimens studied, and n = number of spores measured from given number of specimens. A field Emission Scanning Electron Microscope (FESEM) Hitachi SU-8010 (Hitachi, Ltd., Tokyo, Japan) was used to photograph the morphology of the basidiospores. Sections were studied at magnifications up to 1500× following Sun et al. [18]. 2.2. Molecular Study and Phylogenetic Analysis A CTAB rapid plant genome ex traction kit DN14 (Aidlab Biotechnologies, Beijing, China) was used to acquire total genomic DNA from dried specimens according to the manufacturer’s instructions with some modifications [20,21]. The primer pairs ITS5/4 and MS1/MS2 were used to amplify ITS and the small subunit of mitochondrial rRNA gene (mtSSU) for one-way. The primer pairs LR0R/LR7, NS1/NS4, AF/Cr and 5F/7Cr were used to amplify the large subunit of nuclear ribosomal RNA gene (nLSU), the small subunit of nuclear ribosomal RNA gene (nSSU), DNA-directed RNA polymerase II subunit 1 (RPB1) and DNA-directed RNA polymerase II subunit 2 (RPB2) respectively [22] for two-way. Forests 2021, 12, 932 3 of 17 The PCR procedure for ITS and mtSSU was as follows: initial denaturation at 95 ◦C for 3 min, followed by 35 cycles at 94 ◦C for 40 s, 56 ◦C for 45 s and 72 ◦C for 1 min and a final extension of 72 ◦C for 10 min. The PCR procedure for nrLSU and nrSSU was as follows: initial denaturation at 94 ◦C for 1 min, followed by 35 cycles at 94 ◦C for 30 s, 50 ◦C for 1 min and 72 ◦C for 1.5 min and a final extension of 72 ◦C for 10 min. The PCR procedure for RPB1 and RPB2 was as follows: initial denaturation at 94 ◦C for 2 min, 9 cycles at 94 ◦C for 45 s, 60 ◦C for 45 s, followed by 36 cycles at 94 ◦C for 45 s, 53 ◦C for 1 min, 72 ◦C for 1.5 min and a final extension of 72 ◦C for 10 min. The PCR products were purified and sequenced in Beijing Genomics Institute, China, with the same primers. All newly generated sequences were submitted to GenBank (Table1). Table 1. A list of species, specimens and GenBank accession numbers of sequences used in this study. GenBank Accession No. Species Specimen No. Locality ITS nrLSU nuSSU mtSSU RPB1 RPB2 Phellodon alboniger REB-70 USA KC571749 - - - - - P. alboniger REB-57 USA JN135206 P. atratus CL-72 Canada MK281471 - - - - - P. atratus DAVFP28189 Canada HQ650766 - - - - - MZ225636 P. atroardesiacus Cui 18449 China MZ221189 MZ225598 --- MZ225637 P. atroardesiacus Cui 18457 China MZ225577 MZ225599 MZ225637 --- P. atroardesiacus Cui 18458 China MZ225633 MZ225600 MZ225638 --- P. atroardesiacus Cui 18459 China MZ225634 MZ225601 MZ225639 --- P. atroardesiacus Cui 16951 China MZ225632 MZ225597 MZ225635 - MZ343209 MZ343197 P. brunneoolivaceus REB-166 USA KC571752 - - - - - P. cinereofuscus Cui 14231 China MZ225579 ----- P. cinereofuscus Cui 16940 Australia MZ225580 MZ225602 MZ225640 MZ225623 MZ343210 MZ343198 P. cinereofuscus Cui 16944 China MZ225581 MZ225603 MZ225641 MZ225624 MZ343211 MZ343199 P.